Sialic Acid Derivatives

ABSTRACT

An amine or hydrazide derivative of a sialic acid unit, e.g. in a polysaccharide, is reacted with a bifunctional reagent at least one of the functionalities of which is an ester of N-hydroxy succinimide, to form an amide or hydrazide product. The product has a useful functionality, which allows it to be conjugated, for instance to proteins, drugs, drug delivery systems or the like. The process is of particular utility for derivatising amine groups introduced in sialic acid terminal groups of polysialic acids.

This application is a continuation that claims priority pursuant to 35U.S.C. 120 to U.S. patent application Ser. No. 13/647,326, filed Oct. 8,2012, a continuation application that claims priority to U.S. patentapplication Ser. No. 12/987,878, filed Jan. 10, 2011, now U.S. Pat. No.8,293,888, a divisional application that claims priority to U.S. patentapplication Ser. No. 11/660,128, filed Jul. 13, 2007, now U.S. Pat. No.7,875,708, a 371 national phase filing of PCT/GB2005/003160, filed Aug.12, 2005, that claims priority to European Patent Application EP05251015.3, filed Feb. 23, 2005 and claims priority to InternationalPatent Application PCT/GB2004/003488, filed Aug. 12, 2004, each of whichis incorporated by reference in its entirety.

The present invention relates to derivatives of sialic acid compounds,preferably polysaccharides which have terminal or intrachain sialic acidunits. Preferably the polysaccharide consists only of sialic acid units,for instance linked alpha-2,8, 2,9 to one another. The products areuseful for conjugation to substrates such as peptides, proteins, drugs,drug delivery systems, viruses, cells, microbes, synthetic polymers etc.The reaction involves conjugation of an NHS group containing reagentwith either an amino or hydrazide functional sialic acid derivative.

Polysialic acids (PSAs) are naturally occurring unbranched polymers ofsialic acid produced in certain bacterial strains and in mammals incertain cells [Roth et. al., 1993]. They can be produced in variousdegrees of polymerisation: from n=about 80 or more sialic acid residuesdown to n=2 by either limited acid hydrolysis, digestion withneuraminidases or by fractionation of the natural, bacterial or cellderived forms of the polymer. The composition of different PSAs alsovaries such that there are homopolymeric forms i.e. the alpha-2,8-linkedPSA comprising the capsular polysaccharide of E. coli strain K1 and ofthe group-B meningococci, which is also found on the embryonic form ofthe neuronal cell adhesion molecule (N-CAM). Heteropolymeric forms alsoexist, such as the alternating alpha-2,8 alpha-2,9 PSA of E. coli strainK92 and the group C polysaccharides of N. meningitidis. In addition,sialic acid may also be found in alternating copolymers with monomersother than sialic acid such as group W135 or group Y of N. meningitidis.PSAs have important biological functions including the evasion of theimmune and complement systems by pathogenic bacteria and the regulationof glial adhesiveness of immature neurons during foetal development(wherein the polymer has an anti-adhesive function) [Muhlenhoff et. al.,1998; Rutishauser, 1989; Troy, 1990, 1992; Cho and Troy, 1994], althoughthere are no known receptors for PSAs in mammals. The alpha-2,8-linkedPSA of E. coli strain K1 is also known as ‘colominic acid’ and is used(in various lengths) to exemplify the present invention.

The alpha-2,8 linked form of PSA, among bacterial polysaccharides, isuniquely non-immunogenic (eliciting neither T-cell or antibody responsesin mammalian subjects) even when conjugated to immunogenic carrierprotein, which may reflect its existence as a mammalian (as well as abacterial) polymer. Shorter forms of the polymer (up to n=4) are foundon cell-surface gangliosides, which are widely distributed in the body,and are believed to effectively impose and maintain immunologicaltolerance to PSA. In recent years, the biological properties of PSAs,particularly those of the alpha-2,8 linked homopolymeric PSA, have beenexploited to modify the pharmacokinetic properties of protein and lowmolecular weight drug molecules [Gregoriadis, 2001; Jain et. al., 2003;U.S. Pat. No. 5,846,951; WO-A-0187922]. PSA derivatisation of a numberof therapeutic proteins including catalase and asparaginase [Fernandesand Gregoriadis, 1996 and 1997] gives rise to dramatic improvements incirculation half-life, its stability and also allows such proteins to beused in the face of pre-existing antibodies raised as an undesirable(and sometimes inevitable) consequence of prior exposure to thetherapeutic protein [Fernandes and Gregoriadis, 2001]. In many respects,the modified properties of polysialylated proteins are comparable toproteins derivatised with polyethylene glycol (PEG). For example, ineach case, half-lives are increased, and proteins and peptides are morestable to proteolytic digestion, but retention of biological activityappears to be greater with PSA than with PEG [Hreczuk-Hirst et. al.,2002]. Also, there are questions about the use of PEG with therapeuticagents that have to be administered chronically, as PEG is only veryslowly biodegradable [Beranova et. al., 2000] and both high and lowmolecular weight forms tend to accumulate in the tissues [Bendele, et.al., 1998; Conyers, et. al., 1997]. PEGylated proteins have been foundto generate anti PEG antibodies that could also influence the residencetime of the conjugate in the blood circulation [Cheng et. al., 1990].Despite the established history of PEG as a parenterally administeredpolymer conjugated to therapeutics, a better understanding of itsimmunotoxicology, pharmacology and metabolism will be required [Hunterand Moghimi, 2002; Brocchini, 2003]. Likewise there are concerns aboutthe utility of PEG in therapeutic agents that require high dosages, (andhence ultimately high dosages of PEG), since accumulation of PEG maylead to toxicity. The alpha 2,8 linked PSA therefore offers anattractive alternative to PEG, being an immunologically ‘invisible’biodegradable polymer which is naturally part of the human body, andthat can degrade, via tissue neuraminidases, to sialic acid, a non-toxicsaccharide.

Our group has described, in previous scientific papers and in grantedpatents, the utility of natural PSAs in improving the pharmacokineticproperties of protein therapeutics [Gregoriadis, 2001; Fernandes andGregoriadis, 1996, 1997, 2001; Gregoriadis et. al., 1993, 1998, 2000;Hreczuk-Hirst et. al., 2002; Mital, 2004; Jain et. al., 2003, 2004; U.S.Pat. No. 5,846,951; WO-A-0187922]. Now, we describe new derivatives ofPSAs, which allow new compositions and methods of production ofPSA-derivatised proteins (and other forms of therapeutic agents). Thesenew materials and methods are particularly suitable for the productionof PSA-derivatised therapeutic agents intended for use in humans andanimals, where the chemical and molecular definition of drug entities isof major importance because of the safety requirements of medical ethicsand of the regulatory authorities (e.g. FDA, EMEA).

Methods have been described previously for the attachment ofpolysaccharides to therapeutic agents such as proteins [Jennings andLugowski, 1981; U.S. Pat. No. 5,846,951; WO-A-01879221. Some of thesemethods depend upon chemical derivatisation of the ‘non-reducing’ end ofthe polymer to create a protein-reactive aldehyde moiety (FIG. 1). Thereducing end of PSA (and other polysaccharides) is only weakly reactivewith proteins under the mild conditions necessary to preserve proteinconformation and the chemical integrity of PSA during conjugation. Thesialic acid unit, at the non-reducing terminal of PSA which contains avicinal diol, can be readily (and selectively) oxidised with periodateto yield a mono-aldehyde derivative. This derivative is much morereactive towards proteins and comprises of a suitably reactive elementfor the attachment of proteins via reductive amination and otherchemistries. We have described this previously in U.S. Pat. No.5,846,951 and WO-A-0187922. The reaction is illustrated in FIG. 1 inwhich:

-   -   a) shows the oxidation of CA (alpha-2,8 linked PSA from E. coli)        with sodium periodate to form a protein-reactive aldehyde at the        non-reducing end of the terminal sialic acid and    -   b) shows the reaction of the aldehyde with a primary amine group        of a protein followed by the selective reduction of the Schiff's        base with sodium cyanoborohydride (NaCNBH₃) to form a stable        irreversible covalent bond with the protein amino group.

In PCT/GB04/03488 we describe polysaccharide derivatives which have asulfhydryl-reactive group introduced via a terminal sialic acid unit.This unit is usually introduced by derivatisation of a sialic acid unitat the non-reducing end of the polysaccharide. The sulfhydryl reactivegroup is preferably a maleimido group. The reaction to introduce thisgroup may involve the reaction of a heterobifunctional reagent having asulfhydryl-reactive group at one end and a group such as a hydrazide oran ester at the other end, with an aldehyde or amine group on the sialicacid derived terminal unit of the polysaccharide. The product is usefulfor site specific derivatisation of proteins, e.g. at Cys units orintroduced sulfhydryl groups.

Although the various methods that have been described to attach PSAs totherapeutic agents [U.S. Pat. No. 5,846,951; WO-A-01879221, aretheoretically useful, achievement of acceptable yields of conjugate viareaction of proteins with the non-reducing end (aldehyde form) of thePSA requires reaction times that are not conducive to protein stabilityat higher temperature (e.g. interferon alpha-2b). Secondly, reactantconcentrations (i.e. polymer excess) are required that may beunattainable or uneconomical.

In the invention there is provided a new process for forming derivativesof a sialic acid compound in which a starting compound comprising aterminal sialic acid unit is subjected to a preliminaryintermediate—forming step, in which a group selected from a primaryamine group, a secondary amine group and a hydrazine is formed on theterminal sialic acid unit, followed by a reaction step in which theintermediate is reacted with a bifunctional reagent

in which R is H or sulphonyl; R¹ is a linker group; and X is afunctional group, whereby the ester group is cleaved and the amine orhydrazine group of the intermediate is acylated by —CO—R¹—X to form thederivative.

In a first embodiment the starting compound has a terminal sialic acidunit joined to another moiety via its 2-carbon atom i.e. as anon-reducing terminal unit, and in which the preliminary step involvesoxidation of the C-7, C-8 diol group of the sialic acid to form analdehyde group followed by reductive amination with H₂NR⁴, in which R⁴is H or lower alkyl, or acid addition salt thereof to form theintermediate. This preliminary step is shown in FIG. 3.

In this first embodiment the starting compound has the followingformula:

in which R² is the said other moiety and is selected from a mono-, di-,oligo- or poly-saccharide group, a protein or peptide, a lipid, a drugand a drug delivery system (such as a liposome) and in which the amidederivative product has the following formula:

in which X, R¹ and R⁴ are the same groups as in the respective startingcompounds and R³ is the same as R² or is the product of the reactionthereof in the steps of oxidation, reductive amination and reaction withreagent I. The formation of a compound according to this embodiment isshown in FIG. 6, wherein the reagent I is a bis-NHS crosslinker.

In a second embodiment the starting compound has a reducing terminalsialic acid, joined to another moiety via its 8-carbon atom, and inwhich the preliminary step involves a ketal ring-opening reduction stepwhereby a group having vicinal diols is formed followed by a selectiveoxidation step in which the vicinal diol group is oxidised to analdehyde group, followed by reductive amination with H₂NR⁴ or acidaddition salt to form the intermediate. In this embodiment the startingcompound has the following formula

in which R⁵ is the said other moiety and is selected from a saccharidegroup an oligo- or poly-saccharide group, an alkyl group, an acyl group,a lipid, a drug delivery system, and in which the amide product has thefollowing formula:

in which R¹, X and R⁴ are the same groups as in the respective startingcompounds and R⁶ is the same as R⁵ or is the product of the reactionthereof in the steps of reduction, oxidation, amination and reactionwith reagent I. The formation of a compound of formula V is shown inFIG. 2.

In a third embodiment the starting compound has a terminal sialic acidunit joined to another moiety via its 2-carbon atom (i.e. as anon-reducing terminal unit), and in which the preliminary step involvesoxidation of the C-7, C-8-diol group of the sialic acid to form analdehyde group followed by reaction with hydrazine and reduction to formthe intermediate. In this embodiment in which the starting compound hasthe following formula:

in which R² is the said other moiety and is selected from a mono-, di-,oligo- or poly-saccharide group, a protein or peptide, a lipid, a drugor a drug delivery system and in which the product derivative has thefollowing formula

in which X and R¹ are the same as in the respective starting materialsand R³ is the same as R² or is the product of the reaction thereof inthe steps of oxidation, reaction with hydrazine, reduction and reactionwith reagent I.

In a fourth embodiment the starting compound has a reducing end terminalsialic acid, joined to another moiety via its 8-carbon atom, and inwhich the preliminary step involves a ketal ring-opening reduction stepwhereby a group having vicinal diols is formed followed by a selectiveoxidation step in which the vicinal diol group is oxidised to analdehyde group, followed by reaction with hydrazine and reduction toform the intermediate. In this embodiment in which the starting compoundhas the following formula

in which R⁵ is the said other moiety and is selected from a mono-, di-,oligo- and poly-saccharide group, an alkyl group, an acyl group, a lipidand a drug delivery system, and in which the product derivative has thefollowing formula

in which X, R¹ are same groups as in the respective starting compoundsand in which R⁶ is the same as R⁵ or is the product of the reactionthereof in the steps of reduction, oxidation, reaction with hydrazine,reduction and reaction with reagent I. An example of a reaction schemewhich produces compounds of formula IX is shown in FIG. 5, wherein thebifunctional reagent I is bis-NHS.

In the process it is generally important that the intermediate isisolated substantially from the product mixture of the preliminary stepprior to being contacted with the reagent of formula I. This is becausethe reagents used in the preliminary step(s) may inactivate the reagentof formula I. In addition, where the preliminary step involvessequential steps of oxidation and reduction or vice versa the oxidisingagents or reducing agents of the first step should be inactivated beforeadding the reagent for the subsequent step.

In the process of the invention, it is convenient for the reactionbetween the intermediate and the reagent of formula I to be conducted inan aprotic solvent, preferably comprising a small amount of a proticsolvent. Minimising the level of protic solvent present in the reactionavoids premature deactivation of the NHS group of the reagent of formulaI. In general aprotic solvents are found to damage biological molecules.It is surprising that the use of dimethylsulphoxide DMSO, specificallyto solubilise PSAs, results in good levels of conjugation to NHSreagents, without excess levels of deactivation of the NHS groups priorto reaction, and allows recovery of the derivative from the productmixture. Preferably therefore the aprotic solvent is DMSO.

The reagent of formula I is generally used in an amount which is instoichiometric excess for reaction with the intermediate, and ispreferably present in an amount at least twice, more preferably at leastfive times the amount for stoichiometric reaction with the intermediate.

In one embodiment of the reagent of formula I, X is a group

in which R has the same definition as above.

In an alternative embodiment X is a group selected from the groupconsisting of vinylsulphone, N-maleimido, N-iodoacetamido, orthopyridyldisulfide, protected hydroxyl, protected amino, and azido. The reagentof formula I is preferably selected from:

-   -   N-(α-maleimidoacetoxy)succinimide ester, (AMAS),    -   N-(β-maleimidopropyloxy)succinimide ester, (BMPS),    -   N-(ξ-maleimidocapryloxy)succinimide ester, (EMCS), or its sulfo        analog,    -   N-(γ-maleimidobutyryloxy)succinimide ester, (GMBS), or its sulfo        analog,    -   succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),        (LC-SMCC),    -   m-maleimido benzoyl-N-hydroxysuccinimide ester (MBS), or, its        sulfo analog,    -   succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyate)        (SMCC) or its sulfo analog,    -   succinimidyl-4-(p-maleimido phenyl) butyrate (SMPB) or its sulfo        analog,    -   succinimidyl-6-(β-maleimido-propionamido) hexanoate (SMPH),    -   N-(κ-maleimidoundecanoyloxy) sulfosuccinimide-ester(sulfo-KMUS),    -   succinimidyl 6-[3-2(2-pyridyldithio)-propionamido]hexanoate        (LC-SPDP) or its sulfo analog,    -   4-succinimidyloxycarbonyl-methyl-α-(2-pyridyldithio) toluene        (SMPT) or its sulfo-LC analog,    -   N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),    -   N-succinimidyl[4-vinylsulfonyl)benzoate (SVSB),    -   succinimidyl 3-(bromoacetamido)propionate (SBAP), and    -   N-succinimidyliodoacetate (SIA) and    -   N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) or its sulfo        analog.

Another category of heterobifunctional reagents of formula I havephotoreactive groups as X, such as azide groups. Examples of suchreagents are:

-   -   N-5-Azido-2-nitrobenzoyloxysuccinimide water insoluble        (ANB-NOS),    -   N-Hydroxysuccinimidyl-4-azidosalicylic acid water insoluble,        non-cleavable (NHS-ASA),    -   N-Succinimidyl (4-azidophenyl)-1,3′-dithiopropionate (SADP),    -   Sulfosuccinimidyl        2-(7-azido-4-methyl-coumarin-3-acetamido)ethyl-1,3′-dithiopropionate        (SAED),    -   Sulfosuccinimidyl        2-(m-azido-o-nitro-benzamido)ethyl-1,3′-dithiopropionate (SAND),    -   N-Succinimidyl 6-(4′-azido-2′-nitro-phenylamino)hexanoate        (SANPAH),    -   Sulfosuccinimidyl        2-(p-azido-o-salicylamido)ethyl-1,3′-dithiopropionate (SASD),    -   Sulfosuccinimidyl-(perfluoroazidobenzamido)ethyl-1,3′-dithiopropionate        (SFAD), and    -   N-Hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB).

The reagent of formula I may be selected frombis[2-succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) and its sulfoanalog,

-   -   bis(sulfosuccinimidyl)suberate) (BS³),    -   disuccinimidyl glutarate (DSG),    -   dithiobis (succinimidyl propionate) (DSP),    -   disuccinimidyl suberate (DSS),    -   disuccinimidyl tartrate (DST) or its sulfo analog,    -   3,3′-dithiobis (sulfosuccinimidyl propionate) (DTSSP), and    -   ethylene glycol bis(succinimidyl succinate) (EGS) and its sulfo        analog.

The group R¹ is a difunctional organic radical. Preferably, R¹ isselected from the group consisting of alkanediyl, arylene, alkarylene,heteroarylene and alkylheteroarylene, any of which may substitutedand/or interrupted by carbonyl, ester, sulfide, ether, amide and/oramine linkages. Particularly preferred is C₃-C₆ alkanediyl. Mostpreferably, R¹ corresponds to the appropriate portion of one of thepreferred reagents I listed above. The substituent group may be chosenfrom those listed for R¹ above, or alternatively may be an amino acidside chain.

In the process preferably the product derivative is isolatedsubstantially completely from any excess reagent.

Reaction conditions for the reactions generally used may also be usedhere, for instance with reference to Hermanson, (1995).

More preferably, the product amide derivative is isolated substantiallycompletely from the product mixture. Such isolation and recovery mayinvolve a drying step preferably carried out under reduced pressure andmost preferably a freeze-drying step.

Thus reactive sialic acid derivatives useful for subsequent reactionwith biologically useful compounds may be made available in a stableform.

The invention is illustrated further in the accompanying examples andFigures.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings:

FIG. 1 a is a reaction scheme showing the prior art activation of thenon-reducing sialic acid terminal unit; a) oxidation of colominic acid(a form of polysialic acid) with sodium periodate to form aprotein-reactive aldehyde at the non-reducing end.

FIG. 1 b is a reaction scheme showing the prior art reductive aminationof the aldehyde moiety of reaction scheme 1 a using a protein-aminemoiety; b) selective reduction of the Schiff's base withcyanoborohydride to form a stable irreversible covalent bond with theprotein amino group.

FIG. 2 shows the preparation of reducing and derivatised NHS colominicacid (when non-reducing end has no vicinal diol);

FIG. 3 shows the preparation of reducing end derivatised NH₂-CAcolominic acid (vicinal diol removed at non-reducing end). Specifically,in solution, the terminal sialic acid residue at the reducing end ofpolysialic acid exists in a tautomeric equilibrium. The ketal form,although in low abundance in the equilibrium mixture (Jennings andLugowski, 1981) is weakly reactive with protein amine groups, and cangive rise to covalent adducts with proteins in the presence of sodiumcyanoborohydride, although at a rate and to an extent that are notpractically useful.

FIG. 4 shows the general scheme for preparation of CA-NHS-proteinconjugation;

FIG. 5 shows the preparation of CA-protein conjugates via NHS onreducing end;

FIG. 6 shows preparation of non-reducing end derivatised CA;

FIG. 7 shows the preparation of CA-protein conjugates usingbis(sulfosuccinimidyl) suberate (BS³) on non-reducing end. FIG. 7A showsthe preparation of dervatised CA-SH at the non-reducing end. FIG. 7Bshows CA-protein conjugation using NHS-maleimide. FIG. 7C shows thepreparation of CA-protein conjugates using NHS-maleimide AMAS.

FIG. 8 shows the schematic representation of CA-protein conjugationusing the crosslinker DSG. FIG. 8A shows the preparation of CA-proteinconjugates via NHS on the reducing end. FIG. 8B shows the capping ofreducing end of polysialic acid. FIG. 8C shows the preparation ofnon-reducing end derivatised CA.

FIG. 9 shows the HPLC of the CA-GH conjugation reactions;

FIG. 10 shows the sodium dodecyl sulphate (SDS)-polyacrylamide geleletrophoresis (PAGE) of CA-NHS-GH conjugates (CA 35 kDa);

FIG. 11 shows native PAGE of unreacted CAs. FIG. 11 A shows RI scan onGPC of unreacted 35 kDa CA-N H₂. FIG. 11 B shows RI scan on GPC of 35kDa CA-N H₂ reacted with BS³.

FIG. 12 shows the SDS-PAGE of CAM-β-gal and CAI-β-gal conjugates;

FIG. 13 shows the SDS-PAGE analysis of the CAH-NHS reactions; and

FIG. 14 shows the size exclusion chromatography analysis of the CAH-NHSreactions.

EXAMPLES

Materials

Sodium meta-periodate and molecular weight markers were obtained fromSigma Chemical Laboratory, UK. The CAs used, linear alpha-(2,8)-linkedE. coli K1 PSAs (22.7 kDa average, polydispersity (p.d.) 1.34; 39 kDap.d. 1.4; 11 kDa, p.d. 1.27) were from Camida, Ireland. Other materialsincluded 2,4 dinitrophenyl hydrazine (Aldrich Chemical Company, UK),dialysis tubing (3.5 kDa and 10 kDa cut off limits (MedicellInternational Limited, UK); Sepharose SP HiTrap, PD-10 columns(Pharmacia, UK); XK50 column (Amersham Biosciences, UK); Sepharose Q FF(Amersham Biosciences); Tris-glycine polyacrylamide gels (4-20% and16%), Tris-glycine sodium dodecylsulphate running buffer and loadingbuffer (Novex, UK). Deionised water was obtained from an Elgastat Option4 water purification unit (Elga Limited, UK). All reagents used were ofanalytical grade. A plate reader (Dynex Technologies, UK) was used forspectrophotometric determinations in protein or CA assays.

Methods Protein and CA Determination

Quantitative estimation of CAs, as sialic acid, was carried out by theresorcinol method [Svennerholm 1957] as described elsewhere [Gregoriadiset. al., 1993; Fernandes and Gregoriadis, 1996, 1997]. GH was measuredby the bicinchoninic acid (BCA) colorimetric method.

Reference Example 1 Fractionation of CA by IEC (CA, 22.7 kDa, pd 1.34)

An XK50 column was packed with 900 ml Sepharose Q FF and equilibratedwith 3 column volumes of wash buffer (20 mM triethanolamine; pH 7.4) ata flow rate of 50 ml/min. CA (25 grams in 200 ml wash buffer) was loadedon column at 50 ml/min via a syringe port. This was followed by washingthe column with 1.5 column volumes (1350 ml) of washing buffer.

The bound CA was eluted with 1.5 column volumes of different elutionbuffers (Triethanolamine buffer, 20 mM pH 7.4, with 0 mM to 475 mM NaClin 25 mM NaCl steps) and finally with 1000 mM NaCl in the same buffer toremove all residual CA and other residues (if any).

The samples were concentrated to 20 ml by high pressure ultrafiltrationover a 5 kDa membrane (Vivascience, UK). These samples were bufferexchanged into deionised water by repeated ultrafiltration at 4° C. Thesamples were analysed for average molecular weight and other parametersby GP) and native PAGE (stained with alcian blue). Narrow fractions ofCA produced using above procedure were oxidised with sodium periodateand analysed by GPC and native PAGE for gross alteration to the polymer.

Reference Example 2 Activation of CA

Freshly prepared 0.02 M sodium metaperiodate (NaIO₄; 6 fold molar excessover CA) solution was mixed with CA at 20° C. and the reaction mixturewas stirred magnetically for 15 min in the dark (as shown in the firststep of FIG. 3). The oxidised CA was precipitated with 70% (finalconcentration) ethanol and by centrifuging the mixture at 3000 g for 20minutes. The supernatant was removed and the pellet was dissolved in aminimum quantity of deionised water. The CA was again precipitated with70% ethanol and then centrifuged at 12,000 g. The pellet was dissolvedin a minimum quantity of water, lyophilized and stored at −20° C. untilfurther use.

Reference Example 3 Determination of the Oxidation State of CA andDerivatives

Quantitative estimation of the degree of CA oxidation was carried outwith 2,4 dinitrophenylhydrazine (2,4-DNPH), which yields sparinglysoluble 2,4 dinitrophenyl-hydrazones on interaction with carbonylcompounds. Non-oxidised (CA) and oxidised CA (CAO) (5 mg each) wereadded to the 2,4-DNPH reagent (1.0 ml), the solutions were shaken andthen allowed to stand at 37° C. until a crystalline precipitate wasobserved [Shriner et. al., 1980]. The degree (quantitative) of CAoxidation was measured with a method [Park and Johnson, 1949] based onthe reduction of ferricyanide ions in alkaline solution to ferricferrocyanide (Persian blue), which is then measured at 630 nm. In thisinstance, glucose was used as a standard.

Reference Example 4a Preparation of Amino Colominic Acid (CA-NH₂)

CAO produced as in Reference Example 2 at 10-100 mg/ml was dissolved in2 ml of deionised water with a 300-fold molar excess of NH₄Cl, in a 50ml tube and then NaCNBH₄ (5 M stock in 1 N NaOH(aq)), was added at afinal concentration of 5 mg/ml (FIG. 4, first step). The mixture wasincubated at room temperature for 5 days. A control reaction was alsoset up with CA instead of CAO. Product colominic acid amine derivativewas precipitated by the addition of 5 ml ice-cold ethanol. Theprecipitate was recovered by centrifugation at 4000 rpm, 30 minutes,room temperature in a benchtop centrifuge. The pellet was retained andresuspended in 2 ml of deionised water, then precipitated again with 5ml of ice-cold ethanol in a 10 ml ultracentrifuge tube. The precipitatewas collected by centrifugation at 30,000 rpm for 30 minutes at roomtemperature. The pellet was again resuspended in 2 ml of deionised waterand freeze-dried.

Reference Example 4b Assay for Amine Content

The TNBS (picrylsulphonic acid, i.e. 2,4,6-tri-nitro-benzene sulphonicacid) assay was used to determine the amount of amino groups present inthe product [Satake et. al., 1960].

In the well of a microtitre plate TNBS (0.5 μl of 15 mM TNBS) was addedto 90 μl of 0.1 M borate buffer pH 9.5. To this was added 10 μl of a 50mg/ml solution of CA-amide the plate was allowed to stand for 20 minutesat room temperature, before reading the absorbance at 405 nm. Glycinewas used as a standard, at a concentration range of 0.1 to 1 mM. TNBStrinitrophenylates primary amine groups. The TNP adduct of the amine isdetected.

Testing the product purified with a double cold-ethanol precipitationusing the TNBS assay showed close to 90% conversion.

Example 1 Preparation of CA-NHS

CA-NH2 (35 kDa) (15-20 mg) synthesised in Reference Example 4a above wasdissolved in 0.15 M PBS (350 μL, pH 7.2) and then either 50 or 75 molarequivalents of BS³ in PBS (150 μL, PH 7.2) was added. The mixture wasvortexed for 5 seconds and then reacted for 30 minutes at 20° C. This isshown generally in FIG. 4, second step, for a homobifunctionalcross-linker and more specifically in FIG. 7 for BS³. The CA-NHS productwas purified by PD-10 column using PBS as eluent (pH 7.2) and usedimmediately for site-specific conjugation to the NH₂ groups in proteinsand peptides. Determination of the CA concentration from the PD 10fractions was achieved by analysing the sialic acid content using theresorcinol assay. The NHS content on the CA polymer was measured by UVspectroscopy by analysing the CA and NHS reaction solution at 260 nm andalso by thin layer chromatography with visualization at 254 nm.

CA-NH₂ (35 kDa) (15-20 mg) synthesised in Example 1 above was eitherdissolved in the minimum amount of water (50-65 μL) to which was addedDMSO (300-285 μL) or in >95% DMSO (350 μL) with the aid of heat(100-125° C.). 75 molar equivalents of DSG in DMSO (150 L) was added tothe CA-NH2 solution, vortexed for 5 seconds and then reacted for 30minutes at 20° C. (FIG. 8). The CA-NHS product was purified either withdioxane precipitation (×2) or by PD-10 column using PBS as eluent (pH7.2) and used immediately for site-specific conjugation to the NH2groups in proteins and peptides. As before determination of the CAconcentration from the PD-10 fractions was measured using the resorcinolassay. The NHS content on the CA polymer was measured by UV spectroscopy(260 nm) and by thin layer chromatography (254 nm).

Example 2 Preparation of CA-NHS-Protein Conjugates (Using BS³ and DSG)

GH in sodium bicarbonate (pH 7.4) was covalently linked to CA-NHS (35kDa), from reference example 4b with an excess of BS³. The reaction wasperformed in 0.15 M PBS (pH 7.2; 1.5 ml) using a molar ratio of 25:1 or50:1 of CA-NHS:GH for a period of 30 minutes at 20° C. Polysialylated GHwas characterised by SDS-PAGE and the conjugation yield determined byFPLC-size exclusion chromatography. Controls included subjecting thenative protein to the conjugation procedure using BS³ in the absence ofany CA-NHS. CA-NH2 was also subjected to the conjugation procedure usingBS³ in the absence of native GH.

GH in sodium bicarbonate (pH 7.4) was covalently linked to CA-NHS (35kDa), which was prepared as discussed in example 4b using an excess ofDSG. The reaction was performed in 0.15 M PBS (pH 7.2; 1.5 ml) using amolar ratio of 50:1 of CA-NHS:GH for a period of 30 minutes at 20° C.Polysialylated GH was characterised by SDS-PAGE and the conjugationyield determined by HPLC-size exclusion chromatography. Controlsincluded subjecting the native protein to the conjugation procedureusing DSG in the absence of any CA-NHS.

CA-GH conjugates were dissolved in ammonium bicarbonate buffer (0.2 M;pH 7) and were chromatographed on superose 6 column with detection by UVindex (Agilent, 10/50 system, UK). Samples (1 mg/ml) were filtered over0.45 μm nylon membrane 175 μl injected and run at 0.25 cm/min withammonium bicarbonate buffer as the mobile phase (FIG. 9).

SDS-PAGE (MiniGel, Vertical Gel Unit, model VGT 1, power supply modelConsort E132; VWR, UK) was employed to detect changes in the molecularsize of GH upon polysialylation. SDS-PAGE of GH and its conjugates (withCA-NHS) of 0 (control) and 30 minutes samples from the reaction mixturesas well as a process control (non oxidised CA), was carried out using a4-20% polyacrylamide gel. The samples were calibrated against a widerange of molecular weight markers (FIGS. 10 and 11).

Results

CA and its derivatives (22.7 kDa) were successfully fractionated intovarious narrow species with a polydispersity less than 1.1 with m.w.averages of up to 46 kDa with different % of populations. Table 2 showsthe results of separating the 22.7 kDa material.

TABLE 2 Ion exchange chromatography of CA22.7 (pd 1.3) Elution buffers(in 20 mM Triethanolamine % buffer + mM NaCl, pH 7.4) M.W. Pd Population325 mM 12586 1.091 77.4% 350 mM 20884 1.037 3.2% 375 mM 25542 1.014 5.0%400 mM 28408 1.024 4.4%  425 mM* 7.4% 450 mM 43760 1.032 2.3% 475 mM42921 1.096 0.2% *Not done

This process was scalable from 1 ml to 900 ml of matrix with thefractionation profile almost identical at each scale (not all resultsshown).

[The fractionation of larger polymer (CA, 39 kDa, pd 1.4) producedspecies up to 90 kDa. This process can successfully be used for thefractionation of even large batches of the polymer. The results showthat the ion exchange fractions are narrowly dispersed. This isconsistent with the GPC data.]

All narrow fractions were successfully oxidised with 20 mM periodate andsamples taken from different stages of the production process andanalysed by GPC and native PAGE showed no change in the molecular weightand polydispersity.

Quantitative measurement of the oxidation state of CA was performed byferricyanide ion reduction in alkaline solution to ferrocyanide(Prussian Blue) [Park and Johnson, 1949] using glucose as a standard.The oxidized CA was found to have a nearly 100 mol % of apparentaldehyde content as compared to native polymer. The results ofquantitative assay of CA intermediates in the oxidation process usingferricyanide were consistent with the results of qualitative testsperformed with 2,4 dinitrophenylhydrazine which gave a faint yellowprecipitate with the native CA, and intense orange colour with thealdehyde containing forms of the polymer, resulting in an intense orangeprecipitate after ten minutes of reaction at room temperature.

The amination of the polymer was found to be 85% and the CA-NHS waspositive for NHS. Further, the thiol content of the polymer was found tobe 60%

The integrity of the internal alpha-2,8 linked Neu5Ac residues postperiodate and borohydride treatment was analysed by GPC and thechromatographs obtained for the oxidised (CAO), amino CA (CA-NH₂),CA-NHS materials were compared with that of native CA. It was found(FIG. 9) that all CAs exhibit almost identical elution profiles, with noevidence that the various steps give rise to significant fragmentationor crosslinking (in case of CA-NHS) of the polymer chain. The smallpeaks are indicative of buffer salts.

Formation of the CA-GH conjugates was analysed by SEC-HPLC and SDS-PAGE.For the conjugation reaction with DSG the SDS-PAGE showed that there wasno free GH remaining and that the conjugation reaction had gone tocompletion. This was confirmed by SEC-HPLC, whereby the CA-GH conjugateswere eluted before the expected elution time of the free GH (a peak forfree GH was not observed). On the other hand, analysis by SDS-PAGE ofthe conjugation reaction of CA-NH2 to GH using BS³ showed the presenceof free GH, which was confirmed by SEC-HPLC with an elution peak around70 minutes for the free protein. In addition, the SEC-HPLC enable thedegree of conjugation to be determined at 53%.

The results (FIG. 10) show that in the conjugate lanes there are shiftsin the bands which typically indicates an increase in mass indicative ofa polysialylated-GH in comparison to GH. Further, GH conjugates wereseparated into different species by SEC-HPLC.

Example 3 Preparation of Iodoacetate Derivative of CA (CAI)

3.1 Synthesis

To 40 mg colominic acid amine (85 mol % amine) as (described inReference Example 2) dissolved in 1 ml of PBS pH 7.4 was added 5 mg ofN-succinimidyl iodoacetate (SIA). The mixture was left to react for 1 hat 25° C. in the dark, after which excess SIA was removed by gelfiltration over a 5 ml Hightrap™ Desalting column (AP Bioscience) elutedwith PBS. 0.5 ml fractions were collected from the column and samplesfrom each fraction tested for colominic acid content (resorcinol assay)and reactivity with cysteine indicating Iodide (Ellman's Assay).Fractions positive for both iodide and CA were pooled.

3.2 Conjugation of CAI to β-galactosidase

To E. coli β-galactosidase (5.0 mg, 4.3×10⁻⁸ mol) in 1 ml PBS 15 mg CAIwas added (6.59×10-7 mol, 15 molar equiv). The tube was sealed wrappedin foil and the reaction was allowed to proceed at room temperature for1 h whilst gently mixing. The resulting conjugate was analysed by SDSpage and then purified according to accepted protocols to remove freeCAI. Samples were assayed for polymer and protein content as outlinedabove.

Control reactions were carried out with CA as a negative control. Allsamples were analysed for β-gal activity as described below in section3.3.

3.3 Assay for Enzyme Activity

Standards from 60 μg/ml to 3.75 μg/ml of fresh β-galactosidase wereprepared in PBS. Sample of CAM-β-gal were diluted to 60 μg/ml in thesame buffer. Enzyme activity of the conjugates was measured as follows:In a microtitre plate, to 100 μl of sample or standard was added 100 μlof All-in-One β-gal substrate (Pierce). The plate was incubated at 37°C. for 30 min and absorbance read at 405 nm. A calibration curve wasprepared from the standards and the activity of the samples calculatedfrom the equation for the linear regression of the curve.

3.4 Conclusions

Fractions 3-6 were positive for both polymer and iodoacetate and werepooled. The SDS page (4-12% Bis/Tris gel; FIG. 12) showed an increase inapparent molecular mass for samples incubated with the iodoacetamidederivative but not with control polymer. From the protein and polymerassays the conjugation ratio was determined to be 1.63 CAI:1 β-gal.β-gal activity was calculated to be 100.9% for the conjugated sample,compared to the free enzyme.

Example 4 Preparation of Colominic Acid Hydrazide (CAH) 4.1 Synthesis

Our group has described, in previous scientific papers and in grantedpatents, the utility of natural PSAs in improving the pharmacokineticproperties of protein therapeutics [Gregoriadis, 2001; Fernandes andGregoriadis, 1996, 1997, 2001; Gregoriadis et. al., 1993, 1998, 2000;Hreczuk-Hirst et. al., 2002; Mital, 2004; Jain et. al., 2003, 2004;US-A-05846,951; WO-A-0187922]. Now, we describe new derivatives of PSAs,which allow new compositions and methods of production ofPSA-derivatised proteins (and other forms of therapeutic agents). Thesenew materials and methods are particularly suitable for the productionof PSA-derivatised therapeutic agents intended for use in humans andanimals, where the chemical and molecular definition of drug entities isof major importance because of the safety requirements of medical ethicsand of the regulatory authorities (e.g. FDA, EMEA).

50 mg of oxidised colominic acid (19 kDa) was reacted with 2.6 mg ofhydrazine (liquid) in 400 μl of 20 mM sodium acetate buffer, pH 5.5, for2 h at 25° C. The colominic acid was then precipitated with 70% ethanol.The precipitate was redissolved in 350 μl phosphate buffer saline, pH7.4 and NaCNBH₃ was added to 5 mg/ml. The mixture was allowed to reactfor 4 h at 25° C., then frozen overnight. NaCNBH₃ and reaction byproducts were removed by gel permeation chromatography on a PD10 columnpacked with Sephadex G25, using 0.15 M NH₄HCO₃ as the mobile phase. Thefractions (0.5 ml each) were analysed by the TNBS assay (specific toamino groups; described earlier). Fractions 6, 7, 8 and 9 (the voidvolume fractions) had a strong signal, well above the background. Thebackground was high due to the presence of the NH₄ ⁺ ions. Fractions 6,7, 8 and 9 also contained colominic acid. These four fractions, werefreeze dried to recover the CA-hydrazide (CAH).

4.2 Preparation of Colominic Acid NHS (CA-NHS) and ColominicAcid-Protein Conjugates

10 mg of 19 kDa CA-hydrazide were reacted with 9 mg of BS³ in 400 μl ofPBS (pH 7.4) for 30 minutes at room temperature. The reaction mixturewas applied to a PD-10 column packed with Sephadex G25 collecting 0.5 mlfractions. 0.1 mg of BSA was added to each fraction between 5 and 9.After 2 hours at room temperatures the fractions reacted with BSA. Thesesamples were analysed by SDS-PAGE and SEC HPLC.

These fractions have little colominic acid. The colominic acid richfractions (6 and 7) have a protein streak in addition to the bandspresent in the other samples and BSA, which is clear evidence ofconjugation (FIG. 13).

The HPLC chromatogram of fraction 6 shows that there is a big shift inthe retention time for conjugate as compared to free protein confirmingconjugation (FIG. 14 a and b).

The BSA used contains impurities. The BSA peak is at 56 minutes (FIG. 14a).

In addition to peak at 56 minutes, there are larger species which areconjugates. There is a large peak at 80 minutes, which is the NHSreleased from the CA-NHS as it reacts with the protein. This cannot befree BS³ as the CAH was passed through a gel permeation chromatographycolumn, which will have removed. This strongly suggests that an NHSester group was created on the CA molecule (FIG. 14 b).

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1. A compound of formula Ill or formula VIII

wherein (a) X is a functional group of a NHS-ester, vinyl sulfone,N-maleimido, N-iodoacetamido, orthopyridyl disulfide, hydroxy, protectedhydroxyl, amino, protected amino, carboxyl, protected carboxyl, azide orazido; R¹ is a linker; R³ is a monosaccharide, a disaccharide, anoligosaccharide, or a polysaccharide; and R⁴ is hydrogen or C₁₋₄ alkyl;or (b) R¹ is a linker; X is a functional group; and —R¹—X is

R³ is a monosaccharide, a disaccharide, an oligosaccharide, apolysaccharide; and R⁴ is hydrogen or C₁₋₄ alkyl.
 2. A compoundaccording to claim 1, wherein R¹ is alkanediyl, arylene, alkarylene,heteroarylene or alkylheteroarylene, any of which is optionallyinterrupted by a carbonyl linkage, an ester linkage, a sulfide linkage,an ether linkage, an amide linkage and/or an amine linkage.
 3. Acompound according to claim 2, wherein R¹ is C₃-C₆ alkanediyl.
 4. Acompound according to claim 1, wherein R³ or R⁶ as the case may be is anoligosaccharide or a polysaccharide
 5. A compound according to claim 4,wherein the oligosaccharide is an oligosialic acid.
 6. A compoundaccording to claim 4, wherein the oligosaccharide is a polysialic acid.7. The compound according to claim 1, wherein —R¹—X is a member selectedfrom:

wherein R is H or sulfo.
 8. The compound according to claim 2, wherein—R¹—X is a member selected from:

wherein R is H or sulfo.
 9. The compound according to claim 1, wherein—R¹ is substituted alkarylene.